Abstract
Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis.
Highlights
Hyaluronan is synthesized in the inner face of the plasma membrane by three isoforms of hyaluronan synthases (HAS1, 2, and 3), unique enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space [1]
The Green fluorescent protein glucose transporters (GLUT) (GFP)-HAS3 overexpressing cells were detached during processing, which indicates a decreased adhesion as a result of Hyaluronan synthase MMP (HAS) overexpression, a finding in line with previous results [22]
The results showed that GFP-HAS3 expression significantly induced both the area and the amount of dorsal plasma membrane ruffling as compared to noninduced cells (Figure 3(c))
Summary
Hyaluronan is synthesized in the inner face of the plasma membrane by three isoforms of hyaluronan synthases (HAS1, 2, and 3), unique enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space [1]. Active synthesis of hyaluronan enhances plasma membrane dynamics and formation of several types of actin-based plasma membrane protrusions, like filopodia [2], lamellipodia [3], and membrane ruffles [4]. Ruffles are flat plasma membrane folds that use the actinbased machinery for their dynamic reshaping [5]. Two structurally similar but distinct types of ruffling have been reported, depending on their cellular location. Peripheral ruffling is typically associated with lamellipodia formation and migration [8], while dorsal ruffling is connected to macropinocytosis [9] and internalization of growth factor receptors [10]
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